We have begun to look for distant c-myb transcriptional regulators in the region 25-100 kb upstream region. Because of recent developments in the epigenetics field we can now begin to predict the function of regions of chromatin based upon the presence of histones with specific modifications or specific DNA-binding proteins. Promoter regions and enhancers of activated genes are enriched in histone 3, mono-and tri-methylated on lysine 4 (H3K4me1, H3K4me3) and have acetylated histone 3, for example, H3K9Ac. In an attempt to find regulatory sequences in the upstream region of c-myb, chromatin immunopreciptation analyzed by microchip (ChIP-on-chip) was carried out using antibodies specific for these marks and a tiling microarray representing the 600kb region surrounding the c-myb gene (40bp spacing). When an antibody specific for trimethyl-H3K4 was used in an experiment performed in myeloid blast cells, M1, it was found that this mark was strongly enriched in the region corresponding to the Mml1 integration site (25kb upstream of c-myb) as well as at the c-myb gene. Subsequent experiment with anti- H3K9,14Ac antibodies revealed that these regions were also enriched in acetylated histones, confirming that both of these peaks are associated with transcriptional activity or are poised for activity. Interestingly, abundance of H3K4me3 at both of these regions was decreased in M1 cells following IL-6-induced differentiation, which suggests that activity of the region corresponding to Mml1 integration is correlated with the transcriptional state of c-myb gene. Abundance of the H3K4me3 on the control genes was not affected. Changes in H3K27me3 and H3K9me, reported to be associated with transcriptional repression states were not found to largely differ. Next, ChIP-on-Chip with anti-H3K4me3 was performed in the cells lines carrying retrovirus in the Mml1 region. It was shown previously that the steady-state level of c-Myb mRNA and protein did not differ from M1 cells in these cells (Koller et al., 1996). Interestingly, ChIP-on-chip experiment revealed that the H3K4me3 has a different pattern of distribution at c-myb in 3 out of 4 analyzed Mml1 cell lines, compared to control M. The H3K4me3 signal in these cells spread dramatically toward the 3 end of c-myb and the signal was stronger. This data indicated that virus integration at the Mml results in predominant spreading of the active transcription-related histone mark 25 kb down-stream at the c-myb locus. Since the above observations together suggest that the -25 to -35kb region is associated with the transcriptional activity of the c-myb promoter, we decided to look for evidence of physical interaction of this and other regions upstream of c-myb with the promoter of the gene. Using a Chromatin Conformation Capture (3C, chromatin looping) assay we have been able to show that several regions upstream of c-myb, including the region in proximity of -25 to -35kb H3K4me3 peak, interact with the c-myb promoter in myb-expressing cells. All these interactions disappeared if c-myb was down-regulated by IL-6, suggesting that these interactions are important for the activity of c-myb promoter. In addition there was no interaction detected in NIH/3T3 cells, in which c-myb is not expressed. Interactions observed in M1 cells seem to be preserved in cells carrying retrovirus in Mml1. Interestingly, even larger upstream regions seem to participate in the interactions with the c-myb promoter in the latter cell lines. Since CTCF has been shown by many others to be involved in facilitating looping, we carried out ChIP-on-Chip to look for locations of CTCF binding in M1 cell DNA. Prominent CTCF binding sites were discovered at each of the three retrovirus integration sites, MmL1, MmL2 and Mml3. Studies are being performed to determine if these bound CTCFs are indeed involved in looping. In addition, since our experiments so far suggest that there are c-myb transcriptional regulator regions upstream of the gene, future experiments will also aim to determine whether the DNA sequences in regions of acetylation and H3K4 trimethylation actually function as transcriptional enhancers.